Zebrafish Model with LOV Switch
Induced gene expression system is a valuable tool to study biological processes. Optogenetic expression system can use light as inducer to accurately control the time, location and amplitude of gene expression. Creative BioMart developed an optimized photogenetic protocol for zebrafish to realize photoinduced gene expression in zebrafish embryos. The system exhibits rapid and robust induction, minimal toxicity, and can be activated by several different light transmission modes.
Basic Principle
The protocol relies on the el222 optically gated transcription system. El222 is a naturally occurring light responsive transcription factor found in bacterium E. litoralis. El222 contains a lov domain followed by a helix to helix (HTH) DNA binding domain that binds to a regulatory element (usually C120). When irradiated with blue light, dimerization occurs and responds to gene expression downstream of the promoter. By using light as inducer, EL222 zebrafish system can fine regulate gene expression in space and time, thus overcoming the limitations of traditional inducible expression system.
| Photoreceptor |
Erythrobacter litoralis 222 amino acid protein |
| Binding partner |
/ |
| Cofactor |
Flavin mono-nucleotide |
| Source organism |
Erythrobacter litoralis |
| Mode of action |
Homodimerization, DNA binding |
| Excitation wavelength |
450 nm |
| Reversion wavelength |
Dark |
| Excitation time |
Seconds |
| Reversion time |
Seconds |
Construction of Transgenic Zebrafish Strain
We provide transgenic zebrafish embryo construction and analysis services. The following is a brief construction protocol.
| Steps |
Description |
| Zebrafish crossing and embryo collection |
- Maintain separate transgenic zebrafish lines containing the either EL222 transcriptional activator or regulatory element.
- 6-8 adult zebrafish were hybridized from each crossing using standard methods to produce transgenic embryos containing both EL222 and transcriptional regulatory elements.
- Embryo culture and screening.
|
| Global light induction |
Blue light (465 nm) panels were used to provide activated blue light to multiple embryos at one time. |
| Quantitative assessment of induction by qRT-PCR |
The induction of GFP expression was first detected after 30 minutes of light treatment, and reached more than 130 times the induction peak after 3 hours of light treatment. Expression induction can be evaluated by quantitative real-time PCR (QRT PCR) and fluorescence microscopy. |
| Qualitative assessment of induction by fluorescence microscopy |
- Embryos were removed from the light after the required activation duration.
- The embryos were fixed for imaging in glass recessed slides.
- Use the same image acquisition settings for all samples.
- The bright field and fluorescence images were combined after acquisition using image processing software.
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